Limonene

The use of terpenes in insect control emerged from the necessity of repellents less harmful to the environment and health when compared to synthetic compounds (IBRAHIM et al., 2001). Monoterpenes represent one of the largest and more diverse families of natural compounds, and occur in many forms: acyclic, monocyclics, bicyclics, tricyclics and derivatives (CROTEAU, 1987). They can still exist at hydrocarbon forms or oxygenated radicals, such as: aldehydes, alcohols, ketones, esters, ethers and other functional groups (WEIDENHAMER et al., 1993).

The monoterpenes more broadly distributed in the vegetable kingdom are the main component of volatile oils, responsible for the characteristic smell of many plants, like limonene in citric fruits (SCHUTTE, 1984).

Limonene, a hydrocarbon labeled as monocyclic terpene, is a chiral molecule, present at the environment in both enantiomeric forms D(+)–Limonene and L(-)–Limonene (FERRARINI et al., 2008). L-Limonene is found mainly in a variety of trees and herbs such as Mentha spp.; while D-Limonene is the main component of peel oil from Citrus species (DUETZ et al., 2003).

Figure 1: LimoneneExperimental plots of absorbance versus time versus PEG concentration.

Monoterpenes can be toxic to insects by its penetration through the cuticle; respiratory and digestive system (PRATES et al., 1998). There are indications that they act affecting octopamine receptors (KOSTYUKOVSKY et al., 2002). The insecticide effect of limonene was observed with success for insect control in domestic animals (TONELLI, 1987), as well in haematophagous insects, showing a DL50, on mosquitoes’ larvae, of 53.80 ppm after 24h and 32.52 ppm after 48 hours (KASSIR et al., 1989).

D-Limonene is considered a GRAS (Generally recognized as safe) compound, present at fruit juices, ice creams and sodas, as flavoring agent. It has a low toxicity in humans, even after repetitive exposures in a year. It is clinically used to dissolve gallstones containing cholesterol. Due to its neutralizing effect of gastric acid and assistance in normal peristalsis, it has also been used to relieve heartburn and acid reflux. Even after being experimentally proved that D-Limonene on its natural state does not exhibit significant reactions on human skin, the products of its oxidation does (SUN, 2007). Thinking of this, our objective is to produce limonene at the moment of its use, to not present any risks to health.

The limonene features anti-carcinogenic activity, by inhibiting isoprenylation. It acts by inhibiting the interaction of hydrophobic molecules with G protein, responsible for controlling cellular growth, such as p21ras (CROWELL & GOULD, 1994). With proteic isoprenylation the protein docking to the cellular membrane is facilitated, disabling it in a manner that the control of cellular growth is affected. The limonene also acts in others anti-carcinogenic pathways, like in apoptosis induction (TSUDA et al., 2004) and tumor cell differentiation (GOULD, 1997).

Other important feature is that limonene does not have any relations with sunburn, the damage caused by citric oils to the stratum corneum, by exposition to sun, is caused by the presence of furanocoumarins (JUNIOR, 2009).

Limonene Synthase

Limonene synthase is an enzyme of lyases family responsible to catalyze the reaction that produces limonene from geranyl pyrophosphate. The enzyme shows an optimum pH for its activity between 6-7, and its isoelectric point is at pH of 4,35. Its action mechanism requires Mn2+, but can be replaced by Mg2+. When there is one of these ions and geranyl pyrophosphate the enzyme is capable to catalyze the synthesis of D-limonene. Apparently the activity of this enzyme is inhibited by sodium phosphate or sodium pyrophosphate; also by the application of compounds against histidine, cysteine and methionine, suggesting that at least one of these residues are in the catalytic site (RAJAONARIVONY et al., 1992).

This enzyme was produced in Escherichia coli by Colby et al. (1993). However, the production of limonene was detected only with the use of the recombinant enzyme in vitro, not in vivo. The team of Wisconsin University exhibited, on iGEM 2012, a project to produce limonene using the limonene synthase with the amino acid sequence featured by Colby et al (1993). They produced limonene synthase, but not limonene. It is possible that the constitutive expression of the gene promoted interchain interactions, resulting in insoluble proteic clumps. Thus, possibly, a lower transcription rate would allow the process of folding to happen slowly, and this could provide the correct conformation for the protein.

This project aimed the elaboration of a genetic circuit seeking to overcome the problems related to the recombinant expression of limonene synthase and, for last, produce limonene.

Osmotic stresses represent a simple manner to induce the production of chaperones and foldases, which allows a better protein folding. The polyethylene glycol (PEG) represents an important molecule used to induce this condition (BOLEN et al., 2001). Therefore, there is obtained an efficient production control for the correct folding of limonene synthase, being expressed only by the condition of osmotic stress under the regulation of PuspA promoter. Once activated, it accurately coordinates the expression of limonene synthase.

Coupled to the previous process, we also seek to express a group of proteic chaperones, natural from Escherichia coli, to help the correct folding of limonene synthase. To avoid the use of antibiotics, the genetic circuit will be further integrated to the bacterial genome by the Tn7 system using Flip-Flop transposases.

UspA promoter

In order for the protein expression occurs only at favorable conditions, we will use the UspA promoter (universal stress global response regulator). The PuspA comprises a stress responsive promoter region; this response is related to the expression and translation of universal stress protein A, which characterizes an important element on cellular growth control (FAREWELL et al., 1996).

The promoter was selected using neural network prediction systems, such as Neural Network Promoter Prediction, and heuristics systems, like SCOPE and PePPER. As result we obtained sequences that proved the promoter bidirectionality, and could isolate the sense and antisense sequences. Due the requirements of this project, it was not cloned completely, promoting only in one sense of direction.

Plasmolysis

The process of cellular volume retraction caused by the loss of water in a hypertonic medium is known as plasmolysis. In bacteria, the conformational change, eventually, is not noticed externally, in a way that the cellular membrane can lose total contact with the cell wall by the reduction of the cytoplasmic volume.

The process of plasmolysis alters the cellular metabolism. In Escherichia coli the processes of transcription and translation are inhibited severely, leading to the reduction of other activities regarding the genetic material of the bacteria, and phospholipids synthesis, also inhibited, but in a lesser rate (RUBENSTEIN et al., 1970).

Polyethylene glycol 6000

Many osmolytes can be used to induce plasmolysis, whereas, depending on their characteristics, they can interact with in a greater or lesser extent with cellular components. One of the most used osmolyte to induce osmotic stress is the polyethylene glycol (PEG). The PEG has features based on its chemical structure that offers many forms of applications, in diverse areas such as: pharmacy, industry and alimentary (FRENCH et al., 2009). Its chemical composition and tridimensional arrangement gives hydrophilicity and low melting point, both variables according to the chain length. The solubility in water, methanol, ethanol and benzene makes it useful in many methodologies.

The PEG exhibited to be efficient in inducing osmotic stress, mainly because its flexible and hydrophilic features creates high osmotic pressures, and its chemical arrangement make it less likely to interact with other biologic compounds present at manipulated organisms (MONEY et al., 1989).

Process and applications

The proposed process for the plasmolysis induction by polyethylene glycol is promising in many aspects; among them, the conservation of bacteria at ambient temperature, and also to aid in the process of solubilizing heterologous proteins. It happens because in a osmotic stress situation the bacteria, during the process of cytoplasmic reduction, produce diverse compounds capable to protect its proteins, from chaperones to foldases (SUSIN et al., 2006; MAGER et al., 2000; OGANESYAN et al., 2007; GUNASEKERA et al., 2008). They can aid in proper folding of hard expression proteins (PURVIS et al., 2005; SHIMIZU et al., 2013); conditions already demonstrated in our results, by the conciliation between our promoter responsible to stresses and proteins of hard expression, like limonene synthase.

References

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